RESEARCH FUND FOR THE CONTROL OF INFECTIOUS DISEASES

Characterisation of novel anti-HIV/tuberculosis natural product analogues ZW Chen *, G Liu, XF Lu, L Liu, X Zhang, TCK Lau, SKW Tsui, YX Kang, PR Zheng, BJ Zheng

1 2,5 1 1 2 3 4 Key Messages ZW Chen, G Liu, XF Lu, L Liu, X Zhang, TCK Lau, SKW Tsui, 1 YX Kang, 2 PR Zheng, 1 BJ Zheng 1. 10-chloromethyl-11-demethly-12-oxo- 1 AIDS Institute, Department of Microbiology and Research Centre for calanolide A (F18) is an effective antiretroviral Infection and Immunity, Li Ka Shing Faculty of Medicine, The University compound in vitro. of Hong Kong 2 2. F18 remains effective against HIV-1 strains Tsinghua-Peking Center for Life Sciences and Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, that contain various mutations rendering them Tsinghua University, China resistant to non-nucleoside reverse-transcriptase 3 Department of Biology and Chemistry, City University of Hong Kong inhibitors. 4 School of Biomedical Sciences and Centre for Microbial Genomics and Proteomics, The Chinese University of Hong Kong 5 Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Hong Kong Med J 2015;21(Suppl 7):S14-7 RFCID project number: 09080772 * Principal applicant and corresponding author: [email protected]

Introduction resistance profile, these ten variants were assessed in MT-2 cells in the presence of F18, nevirapine (NVP), Non-nucleoside reverse-transcriptase inhibitors efavirenz (EFV), and etravirine (ETR). All mutant (NNRTIs) are one of the key components of viruses were resistant to F18 (Table 1). The mutant antiretroviral drug regimens against human viruses with mutations L100I or Y188H retained immunodeficiency virus type 1 (HIV-1) replication. high sensitivity to the other three NNRTIs. K103N- (+)-calanolide A, which is purified from the containing virus resulted in more than a 350-fold rainforest plant Calophyllum, is an anti-HIV-1/ change in EC to EFV and NVP, but was sensitive to tuberculosis drug. In our previous study, a library 50 ETR, whereas the HIV-1 variant with V106I/WT and of (+)-calanolide A was constructed and one novel T139R showed moderate resistance to EFV, NVP, compound 10-chloromethyl-11- demethly-12-oxo- and ETR. Therefore, F18-resistant HIV-1 mutations calanolide A (F18) with improved antiviral activity occurred in in vitro long-term culture of MT-2 was identified. This study investigated antiviral cells infected with wild-type virus in the presence breadth, drug-resistance profile, and underlying of F18, and these mutations conferred some cross- mechanism of action of F18. resistance to other NNRTIs.

Methods and results Antiviral activity of F18 against NNRTI- This study was conducted from March 2010 to resistant pseudovirus February 2012. To confirm the relevance of these in vitro-induced resistant mutations, a panel of seven NNRTI- In vitro selection of F18-resistant virus resistant site-directed mutagenesis (SDM)- HTLV-1 transformed human T-cell leukaemia generated pseudoviruses for a GHOST (3)-CCR5 4 (MT-2) cells were infected with 10 TCID50 HIV-1NL4-3 cell-based assay was established. Mutations wild-type virus and cultured in 2-fold increasing that were induced by in vitro F18 selection

concentrations of F18 ranging from 0.14 nM to 20 µM were engineered into a clean HIV-1NL4-3R-E-Luc+ for 120 days. Virus replication was monitored based pseudovirus backbone by SDM. Sensitivity of these on syncytium formation. Ten resistant strains were mutant viruses was determined by infection with selected (Table 1). Genotypic analysis showed that GHOST (3)-CCR5 cells in the presence of F18, NVP seven of ten variants contained a single mutation of or (+)-calanolide A, and by measuring the degree amino acid Leu to Ile at position 100 in the RT gene. of inhibition compared with infection with a wild- In addition, mutations of V292I, K103N, Y188H, type virus (Fig 1a). Virus constructed with the minor V106I, T139R, and P225H also emerged in three Y188H mutation displayed a >100-fold higher level other strains following F18 treatment. of resistance to F18 or (+)-calanolide A, compared To confirm their resistance and cross- with NVP, whereas virus containing the dominant

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TABLE 1. Selection scheme and genotypic analysis of the reverse transcriptase of mutant HIV-1 strains that emerged under dose-

escalating treatment of HIV-1NL4-3 wild-type virus with F18

Strain No. (No. F18 concentration at which Mutation(s) in HIV-1 RT* Fold change† of days in cell the selection initiated and culture) the resistant strain selected F18 NVP EFV ETR 1,4,6,7,8,9,10 0 0.14 nM 84 312 nM L100I 128 20 μM L100I >128 1.67 2.05 1.37 2 0 0.14 nM 91 625 nM Y188H/WT 105 2.5 μM Y188H 128 20 μM Y188H >128 3.75 0.004 0.34 3 0 0.14 nM 98 1.25 μM T139R/WT 120 10 μM V106I/WT, T139R 128 20 μM V106I/WT, T139R 121 36.5 18.06 20.15 5 0 0.28 nM 91 1.25 μM P225H 98 2.5 μM K103N/WT, P225H/WT 105 5 μM K103N 112 10 μM K103N, P225H/WT 120 20 μM K103N, P225H/WT, V292I >128 >357 568.45 2.79 * The complete protease and reverse transcriptase genes were sequenced. The predominant mutation is underlined and in bold

† Calculated as the ratio between the EC50 of the compound for the mutant strain and the HIV-1NL4-3 wild-type strain obtained in the

same experiment at the end of culture. EC50 of F18, NVP, EFV, and ETR against HIV-1NL4-3 wild-type virus is 41.0±2.0 nM, 42.0±1.7 nM, 0.1±0.003 nM, and 0.1±0.002 nM, respectively. Each result is the mean for a single experiment conducted in triplicate

mutation (7 of out 10 strains) L100I induced by F18 in clinical use. One of the most prevalent mutations

in vitro resulted in a 30- and 10-fold change in EC50 among antiretrovial-therapy-experienced patients to F18 and (+)-calanolide A, respectively (Table 1). was Y181C, and virus with this mutation was

NVP susceptibility was noted with L100I mutant sensitive to F18 with an EC50 of 1.0 nM and a viruses. The novel mutation T139R found by F18 reduced susceptibility to NVP for more than a 100-

selection in vitro showed cross-resistance to all fold difference in EC50. Double mutant with K103N/ three compounds NVP, F18, and (+)-calanolide A. Y181C, Y188L and V106A/G190A/F227L mutants To a lesser extent, viruses with K101E and K103N were cross-resistant to the antiviral effects of the mutations also displayed cross-resistance to F18, three NNRTIs. NVP, and (+)-calanolide A. Interestingly, virus with double mutations K103N and P225H exhibited high Antiviral activity of F18 in combination with resistance to NVP with more than a 100-fold change some US-FDA-approved antiretrovirals

in EC50, but resistance to F18 and (+)-calanolide Given the unique antiviral features of F18, its A was abolished when compared with the single efficacy when used in combination with each of mutation K103N. eight US-FDA-approved drugs using PBMC assays As cross-resistant mutations may exist among was determined. There were no antagonistic effects various NNRTIs, susceptibility of ten additional for any combinations of F18 plus another drug

HIV-1 NNRTI-resistant SDMs to F18, NVP, against HIV-1NL4-3 infected PBMCs (Table 2). Most and (+)-calanolide A, alone or in combination, combinations showed a slightly or highly synergistic was determined (Fig 1b). These mutations were effect as calculated by the Prichard and Shipman previously reported based on the current NNRTIs MacSynergy II software.

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(a) 100 nevirapine 40 F18 30 (+) - Calanolide A

20 fold change fold 50

EC 10

0

L100I T139I K101E K103N T139R Y188H

K103N/P225H (b)

100 40

30

20 fold change fold 50

EC 10

0

V106A V179D Y181C Y188L G190A P225H

K103N/Y181C V106A/F227L

V106A/G190A/F227LK103N/Y181C/G190A

FIG. Resistance of constructed HIVNL4-3 SDMs to different non-nucleoside reverse-transcriptase inhibitors (NNRTIs): GHOST (3)-CCR5 cells were infected in the presence of NVP, F18, or (+)-calanolide A with NNRTI mutant pseudoviruses constructed by SDM for (a) mutations identified from Table 1, (b) mutations with resistance to different classes of antiretroviral drugs. The average

EC50 fold changes are shown for triplicate experiments.

* EC50 is not reached at the concentration tested

Discussion than (+)-calanolide A. In addition, due to structural One of the major obstacles to the development of similarity, F18 and (+)-calanolide A shared a similar NNRTIs is the extensive cross-resistance within profile against most NNRTI-cross resistant viruses this class of antiretrovirals. F18 displayed a unique (Fig). These two drugs also displayed their unique profile of cross-resistance. In contrast to NVP, F18 characteristics. For example, F18 was a potent remained effective against HIV-1 SDMs containing inhibitor against (+)-calanolide A-induced T139I NNTRI-resistant mutations T139I, Y181C, V179D, mutant. Conversely, (+)-calanolide A inhibited V106A, K103N/P225H, and K103N/Y181C/G190A. F18-induced L100I mutant more effectively. These From our F18-resistance induction study, F18 did results suggest that the major binding moiety of not readily induce NVP-resistant or (+)-calanolide F18 engages HIV-1 RT at a different binding motif A-resistant viruses, but the resultant dominant towards L100 when compared with T139 usage of L100I mutation attributed to F18-resistance, which (+)-calanolide A. In addition, most of the resistant is similar to another (+)-calanolide A stereoisomer– viruses including L100I selected by F18 can be dihydrocostatolide.1 This finding suggests that F18 inhibited by NVP, EFV, and ETR, indicating a low may function more similarly to dihydrocostatolide level of cross-resistance between F18 and these three

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TABLE 2. Antiviral effect of F18 in combination with eight US-FDA-approved antiretroviral compounds against HIV-1NL4-3 in PBMC assays

Drug Mean synergy volume* Antiviral effect Nucleoside reverse transcriptase inhibitors Zidovudine 87.62 Slightly synergistic Lamivudine 17.32 Additive Stavudine 27.71 Additive Didanosine 297.47 Highly synergistic Non-nucleoside reverse transcriptase inhibitors Nevirapine 73.1 Slightly synergistic Efavirenz 4.01 Additive Protease inhibitors Nelfinavir 116.85 Highly synergistic Integrase inhibitors Raltegravir 54.12 Slightly synergistic

* Positive values represent the synergistic interaction between F18 and other antiretroviral drugs

FDA-approved NNRTIs (Table 1). We conclude thank the HKU-UDF and LSK Faculty of Medicine that the drug-resistance profile of F18 is distinctly Matching Fund for financial supports to HKU AIDS different from that of other NNRTIs,2-4 possibly due Institute. to a distinct binding motif of F18 to HIV-1 RT that Results of this study have been published in: Lu differs to other NNRTIs. X, Liu L, Zhang X, et al. F18, a novel small-molecule Since the discovery of NNRTIs, combination nonnucleoside reverse transcriptase inhibitor, therapy of at least three antiretrovirals has become inhibits HIV-1 replication using distinct binding the gold standard for clinical treatment of HIV-1 motifs as demonstrated by resistance selection and patients in the last 20 years. To avoid issues of drug- docking analysis. Antimicrob Agents Chemother drug interaction, we tested the combined anti-HIV 2012;56:341-51. activity of F18 with each of eight commonly used antiretrovirals. As a natural product-derived small References molecule, F18 had no antagonistic effect when used 1. Buckheit RW Jr, White EL, Fliakas-Boltz V, et al. Unique in two-drug combination against both wild-type and anti-human immunodeficiency virus activities of the drug-resistant viruses (Table 2). Interestingly, the nonnucleoside reverse transcriptase inhibitors calanolide lack of antagonistic effects between F18 and NVP A, costatolide, and dihydrocostatolide. Antimicrob Agents or EFV provided further evidence that F18 does Chemother 1999;43:1827-34. not share identical binding motifs to HIV-1 RT with 2. Azijn H, Tirry I, Vingerhoets J, et al. TMC278, a next- either NNRTI. generation nonnucleoside reverse transcriptase inhibitor (NNRTI), active against wild-type and NNRTI-resistant Acknowledgements HIV-1. Antimicrob Agents Chemother 2010;54:718-27. 3. Richman DD, Havlir D, Corbeil J, et al. Nevirapine This study was supported by the Research Fund for resistance mutations of human immunodeficiency virus the Control of Infectious Diseases, Food and Health type 1 selected during therapy. J Virol 1994;68:1660-6. Bureau, Hong Kong SAR Government (#09080772) 4. Vingerhoets J, Azijn H, Fransen E, et al. TMC125 displays and the 11th Five-Year Mega Project on the a high genetic barrier to the development of resistance: prevention and treatment of AIDS, viral hepatitis, evidence from in vitro selection experiments. J Virol and other infectious disease (2009ZX09501-012). We 2005;79:12773-82.

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